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Keywords:

  • leptin;
  • milk;
  • breast-feeding;
  • infants;
  • metabolic imprinting

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Objective: Leptin, a hormone that regulates food intake and energy metabolism, is present in breast milk. The aim of this study was to determine whether milk leptin concentration is correlated with maternal circulating leptin and BMI and with body weight gain of infants.

Research Methods and Procedures: A group of 28 non-obese women (BMI between 16.3 and 27.3 kg/m2) who breast-fed their infants for at least 6 months and their infants were studied. Venous blood and milk samples were obtained from mothers at 1, 3, 6, and 9 months of lactation, and leptin concentration was determined. Infant body weight and height were followed until 2 years of age.

Results: During the whole lactation period, milk leptin concentration correlated positively with maternal plasma leptin concentration and with maternal BMI. In addition, milk leptin concentration at 1 month of lactation was negatively correlated with infant BMI at 18 and 24 months of age. A better negative correlation was also found between log milk leptin concentration at 1 and at 3 months of lactation and infant BMI from 12 to 24 months of age.

Discussion: We concluded that, in a group of non-obese mothers, infant body weight during the first 2 years may be influenced by milk leptin concentration during the first stages of lactation. Thus, moderate milk-borne maternal leptin appears to provide moderate protection to infants from an excess of weight gain. These results seem to point out that milk leptin is an important factor that could explain, at least partially, the major risk of obesity of formula-fed infants with respect to breast-fed infants.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Obesity and overweight are the most frequent nutritional disorders in children and adolescents in industrialized countries, and there is a continuing increase in their prevalence (1). Numerous studies have found an association between being overweight as a child and being overweight in adulthood (2, 3) with the risk of associated health complications such as cardiovascular disease and diabetes (2, 4). Given that obesity is associated with considerable morbidity, is increasing in prevalence, and is often recalcitrant to therapy (5, 6), the identification of strategies for its prevention is of crucial relevance.

Concerning obesity prevention, the notion that nutrition during the early phases of human development can predispose or program individuals to adult disease has aroused considerable interest, particularly since the last decade. The fetal origins hypothesis of Barker et al. (7) proposes that poor fetal nutrition causes adaptations that program future propensity to obesity and other related diseases. Much evidence supports this idea, such as the emblematic example of the Dutch famine (8, 9). Adults who were conceived during the acute famine that ravaged the western part of Holland during the last 6 months of World War II, whose mothers experienced poor nutrition in the 1st and 2nd trimesters of their pregnancies, were more likely to be obese than their peers whose mothers did not experience poor nutrition. In contrast to the contribution of the fetal period, the contribution of the early postnatal environment, particularly early nutrition during lactation, has received less attention. Different studies have shown a clear association between overnutrition during infancy and later obesity (10, 11). In this sense, a vigorous breast-feeding style, with high energy intake, has been associated with greater adiposity in the first 6 years of age (12, 13). In addition, several studies have shown that breast-feeding, compared with formula feeding, is associated with a lower risk of later obesity (14, 15, 16, 17, 18), although some studies do not support this hypothesis (19, 20). The duration of lactation has also been inversely associated with obesity and overweight, although generally to a lesser extent (14, 15, 16, 17). Breast milk is known to contain many bioactive hormones and peptides, which may play important roles in neonatal health and development (21, 22). However, which breast milk components are involved are, as yet, unknown.

Leptin is an anorexigenic hormone (23) that plays an important role in the central regulation of energy balance, decreasing food intake and increasing energy expenditure (24, 25). Leptin is produced mainly by the adipose tissue but also by other tissues such as placenta (26, 27), stomach (28, 29, 30), and mammary epithelium (31), and is also present in maternal milk (32, 33). In rats, it has been shown that leptin present in maternal milk can be absorbed by the immature stomach of nursing rats (32, 34, 35) and transferred to the infant rat circulation (32). Leptin supplied from maternal milk is the main source of leptin in the stomach during the first one-half of the suckling period, whereas endogenous production of leptin by the gastric mucosa increases at the end of the suckling period and with the change of diet to solid food (34). We have shown recently that oral administration of leptin, with doses close to the physiological concentration present in milk, can reduce food intake in suckling rats (35). Thus, exogenous leptin supplied by maternal milk could regulate short-term feeding in neonates and exert other biological effects, at a time in which both the adipose tissue and the appetite regulatory systems are immature (35).

In humans, the role of milk leptin in lactating infants is not known. However, it is known that leptin concentration in human milk varies significantly between people (32, 33), and some authors have found a positive correlation between leptin concentration in milk and maternal plasma leptin concentration and adiposity (33, 36), although contrary data have been published (37). It is not known whether the amount of leptin supplied with maternal milk or the lack of supply when using infant formulas (38) may have significant effects on infant development and whether this may have further implications in the prevention of or propensity toward obesity in adulthood. The present study was aimed at determining, in a group of non-obese mothers who breast-fed their infants during a period of at least 6 months, whether milk leptin concentration is correlated with maternal plasma leptin concentration and BMI and with body weight gain of infants during the first 2 years.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Subjects

Twenty-eight healthy lactating women ages 23 to 37 years (31.19 ± 0.68 years) with a normal BMI of 21.6 ± 0.5 kg/m2 (range, 16.3 to 27.3 kg/m2) who planned to breast-feed their full-term newborn infants for a long period (at least 6 months) were enrolled in the study. Maternal anthropometric parameters, such as height and body weight before gestation and during the lactation period and the evolution of infant body weight and length during the whole lactation period and until 2 years of age, were registered. BMI was determined as weight in kilograms divided by height in meters squared. The protocol followed in this study was reviewed and approved by the Ethical Committee of our Regional Government, and written informed consent was obtained for all participants.

Sample Collection

During the whole lactation period, venous blood and milk samples were obtained from mothers at 1 (±3 days) and 3, 6, and 9 (±1 week) months. To minimize variation in leptin concentrations in plasma due to time of day and time relative to eating a meal, all blood samples were obtained in the morning at 9 to 10 am after an 11- to 12-hour fast. Blood samples were collected in EDTA tubes and then centrifuged at 850 g for 10 minutes at 4 °C to collect the plasma. Milk samples were collected manually in the morning immediately after suckling, when the infant had self-terminated suckling due to satiation. Previously published results have shown no significant difference between leptin levels of breast milk samples obtained in the initial and terminal phases of suckling (37). Breast milk and plasma samples were stored frozen at −20 °C until analysis.

Leptin Assay

Leptin concentration in maternal plasma and in whole milk was determined by a commercially available enzyme-linked immunosorbent assay kit (human leptin; R&D Systems, Minneapolis, MN).

Concerning milk leptin analysis, we previously validated the assay and tested different described protocols for sample preparation (31, 33, 38) and different dilutions with the assay buffer to ensure the suitability of the enzyme-linked immunosorbent assay kit for these samples: whole milk samples (vigorously vortexed to ensure sample uniformity); skimmed milk samples, prepared by centrifuging whole milk at 8000 g for 15 minutes at 4 °C to separate milk fat (31); sonicated milk, prepared by 5-second bursts with 20-second cooling intervals using a Misonix sonicator (33); and lipase-treated milk [obtained by adding 3 μL of pancreatic lipase (Sigma, Madrid, Spain) and 6 μL of 1 M sodium bicarbonate to 600 μL of whole milk samples and incubating them at 37 °C for 1 hour, according to (38)] to degrade triacylglycerides, which could interfere with the immunoassay. In addition, samples were supplemented with 125 ng/L human leptin standard before analysis, and recovery of added leptin was calculated. No significant differences were observed with these different protocols, except by using sonicated samples, where leptin concentration detected was lower; thus, we decided to use whole milk samples diluted 1:1 with the assay buffer. The recovery of added leptin using this protocol was 103.1 ± 1.4% (n = 4).

Statistical Analysis

Simple correlations were assessed by Pearson's correlation coefficients. Student's t test was used to compare mean values of leptin concentration in maternal plasma and in breast milk. In all cases, threshold of significance was defined as p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The characteristics of mothers and infants are shown in Table 1. The mothers included were not obese (BMI before gestation was between 16.3 and 27.3 kg/m2, and the mean was 21.6 ± 0.5 kg/m2), only three of them were slightly over a BMI of 25 (25.3, 25.8, and 27.3 kg/m2), and their infants were born full-term (gestation age, 39.7 ± 0.2 weeks). All infants were breast-fed for at least 6 months (and exclusively for at least 4 months).

Table 1.  Characteristics of lactating mothers and their infants
  1. Data are shown as means ± standard error (range). ND, non-detected values. Maternal BMI value considered was before pregnancy.

Mothers 
Age (years)31.2 ± 0.7 (23 to 37)
 BMI (kg/m2)21.6 ± 0.5 (16.3 to 27.3)
 Plasma leptin concentration at 1 month (ng/mL)12.8 ± 1.7 (6.7 to 37.5)
 Milk leptin concentration at 1 month (ng/mL)0.156 ± 0.039 (ND to 0.853)
Infants 
 Sex (female/male)16/12
 Weight at birth (kg)3.20 ± 0.07 (2.33 to 3.97)
 Weight at 2 years (kg)12.3 ± 0.3 (9.3 to 15.6)
 BMI at 2 years (kg/m2)16.1 ± 0.3 (13.8 to 20.4)

Mean values of breast milk and maternal plasma leptin concentrations are shown in Table 1. In agreement with the literature (32, 33), in our study, the concentration of leptin in breast milk was significantly lower than in maternal plasma (p < 0.001, Student's t test) during the whole lactation period. There was a positive correlation between leptin concentration in milk and in maternal plasma at each of the measured time-points (1, 3, 6, and 9 months), which are shown plotted all together in Figure 1A (r = 0.519, p < 0.001). In addition, maternal BMI correlated positively with plasma leptin concentration (Figure 1B; r = 0.598, p < 0.001) and with milk leptin concentration (Figure 1C; r = 0.387, p < 0.001) at each of the measured time-points. It is remarkable that a stronger correlation was found between maternal log BMI and both maternal plasma and milk log leptin concentration (r = 0.696 and 0.607, respectively) (data not shown).

image

Figure 1. Correlations between leptin concentration in milk and in maternal plasma (A), leptin concentration in maternal plasma and maternal BMI (B), and leptin concentration in milk and maternal BMI (C) throughout the whole lactation period. Plasma and milk samples were collected from 28 mothers at 1, 3, 6, and 9 months. Leptin concentrations were determined as described in “Research Methods and Procedures.” Simple correlations were assessed by Pearson's correlation coefficients. The r and p values for correlations are indicated.

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Milk leptin concentration at 1 month of lactation, but not at any other time-point studied, was negatively correlated with infant BMI at 18 and 24 months of age (r = −0.493, p < 0.05; r = −0.456, p < 0.05, respectively; see Figure 2 A for correlation at 24 months of age). Additionally, a stronger correlation was found between the log-transformed milk leptin concentration at 1 and 3 months of lactation and infant BMI from 12 to 24 months of age; see Figure 2B showing the correlation between log milk leptin concentration at 1 month of lactation and infant BMI at 24 months of age. Correlation values at 1 month of lactation were r = −0.434, p < 0.05; r = −0.547, p < 0.01; and r = −0.601, p < 0.01; at 12, 18, and 24 months of life, respectively; correlation values at 3 months of lactation were r = −0.486, p < 0.05; r = −0.451, p < 0.05; and r = −0.505, p < 0.01; at 12, 18, and 24 months of life, respectively.

image

Figure 2. Correlations between milk leptin concentration (A) or log milk leptin concentration (B) at 1 month of lactation and infant BMI at 2 years of age. Milk leptin concentration was determined as described in “Research Methods and Procedures.” Simple correlations were assessed by Pearson's correlation coefficients. The r and p values for correlations are indicated.

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No significant correlations were found between milk leptin concentration and infant body weight and body weight gain at all ages reported, when all data were considered. It should be noted that most milk leptin concentrations ranged from undetected values (<0.0078 ng/mL in two subjects) to 0.35 ng/mL; however, there were two higher values at 1 month of lactation (0.629 and 0.853 ng/mL) that were above that range. Without considering these two high values, milk leptin concentration at 1 month of lactation, but not at any other time-point studied, was negatively correlated with infant body weight and with body weight gain at all ages reported (1, 3, 6, 9, 12, 18, and 24 months of life) (see correlations for some representative ages in Figure 3).

image

Figure 3. Correlations between milk leptin concentration at 1 month of lactation and infant body weight (left) or body weight gain from birth (right) at 1 (top), 12 (middle), and 24 (bottom) months of age. Milk leptin concentration was determined as described in “Research Methods and Procedures.” Simple correlations were assessed by Pearson's correlation coefficients. The r and p values for correlations are indicated.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

To our knowledge, this study provides the first evidence showing that leptin present in breast milk may regulate body weight gain during infancy in humans and, thus, supports the hypothesis that leptin is one of the bioactive components present in milk that could be responsible for the role of breast-feeding in lowering the risk of childhood obesity.

Leptin concentration in milk varies widely among people (32, 33). Some authors have found a positive correlation between maternal BMI or adiposity and plasma leptin concentration (33, 36), although contrary results have also been published (37). We have studied milk leptin concentration during the lactating period in a group of non-obese mothers and also have found a wide variation, ranging from values under the detection limit of the assay (<0.0078 ng/mL) to 0.853 ng/mL. In addition, we have found that milk leptin concentration positively correlates with both maternal plasma leptin concentration and BMI at any time-point of the lactation period studied. Even considering methodological differences, values obtained in this group of non-obese mothers are lower than values published for women with higher BMI values (33). Thus, the amount of leptin supplied to an infant through breast milk depends on the mother's adiposity. Lean mothers with very low plasma leptin concentrations are producing milk with little or even no significant leptin, similar in this sense to infant formula, which does not have leptin as an ingredient (38). Conversely, only breast-fed infants nursed by mothers with relatively significant adiposity are exposed to significant amounts of leptin in milk.

Although some studies do not support this hypothesis (19, 20), there is increasing epidemiological evidence suggesting that breast-feeding compared with infant formula confers protection against obesity in later life (14, 15, 16, 17, 18), but which components are responsible is currently unknown. Human milk contains many hormones and growth factors (21), the function of some of which is still unknown in neonatal development. The known role of leptin in energy balance (24), its presence in human milk (32, 33), and its absence in infant formula (38) make leptin a good candidate to be considered. However, a correlation between milk leptin concentration and infant body weight had not yet been demonstrated. We show here that, at least in non-obese nursing women, both leptin concentration (at 1 month of lactation) and log milk leptin concentration (mainly at 1 month of lactation but also at 3 months) are negatively correlated with infant BMI at 2 years of age. This correlation was more pronounced considering log milk leptin concentration but lineal correlation was also significant at least in this margin of BMI. This would indicate that differences in milk leptin concentration within the lower range may have a big impact on infant BMI; differences within the range of higher milk leptin concentrations may have less repercussion on infant BMI.

Results also show that the first stages of lactation, a period in which the gastric mucosa is still immature and the absorption of intact leptin may be facilitated (38), are of critical relevance for the action of leptin. Moderate milk-borne maternal leptin mainly during these first stages of neonatal development can cause an adaptation that programs future obesity prevention. Interestingly, the two highest values of infant BMI at 2 years of age (20.4 and 18.1 kg/m2), which are over the 85th percentile, come from two infants who were fed by two mothers whose milk leptin concentration was under the assay detection limit (<0.0078 ng/mL).

These results in infant humans agree with our previous observations in neonate rats. We described that leptin orally taken by neonate rats can reach target cells through the immature stomach (34). In addition, leptin orally administered to neonate rats, in a dose close to physiological concentration present in milk, is absorbed by the stomach and reaches circulation, resulting in lower food intake (35). Milk leptin absorption by the stomach of neonate rats has been particularly observed during the first one-half of the lactation period, when stomach production of leptin is still low (34). During the second one-half of this period and paralleling the maturation process of the gastric mucosa, the absorption of exogenous leptin decreased, whereas endogenous production by gastric mucosa increased (34). The fact that leptin absorption by the immature stomach occurs, at least in rats, mainly during the first stages of the lactation period could explain the importance of the amount of leptin supplied during this period on body weight control in later life.

Leptin receptor is expressed in the vagal nerve innervation of the stomach (39); thus, oral leptin could act either through the activation of afferent vagal nerves (leptin in the gastric lumen) or centrally (leptin in the systemic circulation). Both pathways could participate in the short-term regulation of food intake during the neonatal period (35).

Previous studies have not found a significant association between milk leptin concentration and infant adiposity (36, 37). Several factors such as the time-point of sampling during the lactation period, the shorter period of follow-up of infant weight, and the inclusion of obese women and obese infants in those studies can make the difference. In fact, family heredity, involving genetic and shared environmental components, which are a strong determinant of childhood obesity (40), could be masking the effects of leptin. In our study, we have enrolled a more homogeneous group of non-obese mothers. In addition, we have not considered the effect of high milk-borne maternal leptin, e.g., characteristic of obese or overweight women; however, the two cases of maternal milk with abnormally high leptin concentration in relation to their BMI did not seem to provide further protection against body weight gain.

In conclusion, to our knowledge, this is the first time that, in a population of non-obese mothers, it is shown that infant body weight may be influenced by milk leptin concentration until 2 years of age. Moderate amounts of leptin supplied by normal weighted mothers through the milk seem to provide moderate protection to infants from an excess of weight gain. This protective effect could be sharpened in adulthood. The lower protection observed in very lean mothers seems to point out that milk leptin is an important factor that could explain, at least in part, the major risk of overweight and obesity of formula-fed infants during adulthood with respect to breast-fed infants.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by the Spanish Government (Grants G03/028, BFI2003-04439, and AGL2004-07496/ALI). We acknowledge the women and infants who participated in the study and also the Balearic Association for Breast-feeding, and the midwives C. Artigues and M. Mantxola, who supported mothers during the lactation period.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
Footnotes
  1. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.